1. Field of the Invention
The present invention concerns the field of high throughput assays of molecules. More particularly, the present invention concerns methods and apparatus of use in DNA sequencing and other high throughput assays, using a novel hybrid apparatus comprising an array of capillaries attached to a microfabricated chip injector.
2. Description of Related Art
DNA sequencing chemistry was first developed by Sanger et al. (1977) and by Maxam and Gilbert (1977). Sanger""s dideoxy chain termination method is the most widely used for high-volume sequencing, due to the development of automated fluorescence sequencing based on labeled primers or terminators (Smith et al., 1986; Prober et al., 1987; Tabor et al., 1990; Ansorge et al., 1987). Implementation of this technology has produced automated slab-gel-based sequencers with 1-2 kb/hr capacity (Hunkapiller et al., 1991). This capacity may be pushed to 5 kb/hr through incremental improvement by increasing the number of lanes, decreasing the gel thickness, and improving the labeling chemistry and the detection capabilities. Such improvements will plateau unless revolutionary technique(s) are invented and applied. Generally, the slab gel format is not easily adapted for automated sample loading and it is probably incompatible with further efforts to miniaturize the sequencing process.
A number of advances have been made in DNA sequencing technology since 1990. These originate from two developments. First, by reducing the cross section of the gel cavity, higher electric fields can be applied without producing gel-heating anomalies, thereby providing faster separations. Second, laser-excited fluorescence detection is so sensitive that smaller separation lanes can be easily detected.
A number of workers have explored the use of capillary gel electrophoresis (CGE) for the rapid separation of DNA extension fragments in one-color and eventually 4-color detection formats (Drossman et al., 1990; Luckey et al., 1990; Swerdlow et al., 1991; Cohen et al., 1990; Ruiz-Martinez et al., 1993; Best et al., 1994). Capillary electrophoresis (CE) separations of DNA sequencing fragments are about 10 times faster than slab gels and are often complete in under 2 hr. However, the throughput of CE-based DNA sequencing has been limited by the lack of a method for running multiple capillaries (lanes) in parallel.
Mathies and coworkers (Huang et al., 1992a, 1992b; Mathies et al., 1992) developed a solution to this limitation by using a laser-excited confocal fluorescence scanner (Mathies et al., 1994) to interrogate bundled capillaries. The capillaries were translated past the focused laser beam and sequentially sampled. The capillaries separated at the injection end of the array to facilitate rapid sample loading from a microtitre dish array.
Several different groups have developed alternative capillary array apparatus. Kambara and coworkers (Takahashi et al., 1994) developed a capillary array system based on sheath flow detection scheme. Two different laser excitation beams were passed through the sheath flow and the fluorescence was imaged to a CCD for multicolor detection. This format has the advantage that all lanes are continuously excited and the flow cell has good optical quality with low scattering noise. Disadvantages of this arrangement include the complexity of the sheath flow cell and extra band-broadening due to the electric field distortion in the flow cell. A conceptually similar system was described by Dovichi et al. (1995). Ueno and Yeung (1994) developed a capillary array system where the laser is line-focused on a stationary array and the resulting fluorescence is imaged to a CCD for detection. A two-color-ratio method was used for DNA sequencing. Two 96-capillary-array DNA sequencers have been successfully developed and are currently used. One is based on Mathies"" confocal detection design and the other is based on Kambara""s sheath flow arrangement.
Research efforts have also been given to sequencing with short separation channels on microfabricated CE chips (Woolley et al., 1995; Schmalzing et al., 1998; Liu et al., 1999) or short capillaries (Muller et al., 1998) to increase the separation speed. Woolley and Mathies (1995) performed high-speed separations of DNA sequencing fragments on microfabricated CE chips. DNA separations were achieved in 50 xcexcmxc3x978 xcexcmxc3x973.5 cm channels microfabricated in a 2-in.xc3x973 in. glass sandwich structure. Approximately 150 bases of four-color sequencing mixture were separated in 9 min and base-assigned with an accuracy of 97%. Single-base resolution was obtained out to 200 bases for both the one- and four-color separations. Alternative methods for detection of separated molecules on CE chips have been disclosed (U.S. Pat. No. 5,906,723, incorporated herein by reference in its entirety.)
Theoretically, high-speed separation should be achieved on capillaries provided they are short. Muller et al. (1998) have experimentally demonstrated this using a 7-cm-long (50-xcexcm-i.d.) capillary and one-color sequencing mixture. Single base resolution was observed up to 300 bases in 3 min. However, it is difficult to arrange 96 or more such capillaries to carry out sample injection and separation for high-throughput DNA sequencing. Recently, Schmalzing, et al (1998) performed a separation of one-color sequencing mixture using an 11.5-cm-long microfabricated channel. Single-base resolution was obtained up to xcx9c400 bases in 14 min. Liu et al. (1999) have demonstrated high-speed DNA sequencing on a 7 cm-long microfabricated channel. Single-base resolution of reached 500 bases in 9.2 min using a one-color sequencing mixture, and four-color sequencing exhibited a base-calling accuracy of 99.4% up to 500 bases.
Current efforts on the development of high-speed and high-throughput DNA sequencing are in two major areas, conventional CGE and microfabricated electrophoresis chips. Research with conventional CGE focuses on improving the sieving matrix and separation conditions, and increasing the number of capillaries. Research on microfabricated electrophoresis chips is more exploratory. In order to achieve high-speed and high-throughput analysis, Mathies et al. (1999) developed a radial chip. This chip has a common anode reservoir in the center of a circular 10 cm diameter wafer and an array of 96 channels extending outward toward injector units at the perimeter of the wafer. A rotary scanning detection system consists of a rotating objective head coupled to a four-color confocal detection unit. High-speed and high-throughput assays have been demonstrated on this radial chip for genotyping. High quality sequencing has not been obtained due to the limited effective separation distance.
Other attempts are directed to the manufacture of large xe2x80x9cchipsxe2x80x9d in order to achieve high quality sequencing separation. These approaches gain back the read-length from conventional CGE, but give up the separation speed of microfabricated CE chips. In addition, xe2x80x9cmicroxe2x80x9d fabrication of a half-meter size chip without defects is challenging.
Sequencing separation using short separation channels (Schmalzing et al., 1998; Liu et al., 1999) improves separation speed about 10 fold compared to conventional capillaries (xcx9c40 cm). However, the sequencing read-lengths diminish by a factor of 1.5 to 2.
An unresolved need exists in the art for the development of high speed, high-throughput DNA sequencing methods and apparatus that are capable of reading DNA sequences significantly longer than 500 bases, using small amounts of DNA sample in a small sample volume. None of the methods or apparatus discussed above are capable of such separations.
The present invention solves a long-standing need in the art by providing a hybrid apparatus for high-speed, high throughput and long read length DNA sequencing separation, comprising a microfabricated chip injector attached to an array of one or more capillaries. Within the scope of the invention almost any number of capillaries may be incorporated into the apparatus, from 1, 2, 4, 8, 16, 24, 32, 48, 64, 96, 128, 160, 192, 224, 256, 288, 320, 352, 384, 416, 480, 544, 608, 672, 736, 800, 864, 928, 960 or more capillaries.
In a particularly preferred embodiment, the chip injector is configured as shown in FIG. 1. Each capillary is inserted into a connection channel. Each connection channel is connected to an injector, a cathode reservoir, a sample reservoir and a waste reservoir. Cross channels connect the sample reservoir and waste reservoir to the injector. In preferred embodiments, the inside diameter (i.d.) of the connection channel is fabricated to precisely match the outside diameter (o.d.) of the capillary, while the i.d. of the injector and cross channels is fabricated to precisely match the i.d. of the capillary. In preferred embodiments, the dead volume is less than 2 nanoliters, more preferably less than 1 nanoliter, more preferably less than 500 picoliters, more preferably less than 200 picoliters, more preferably less than 100 picoliters, more preferably less than 50 picoliters, more preferably less than 20 picoliters, more preferably less than 10 picoliters, more preferably less than 5 picoliters, more preferably less than 2 picoliters per capillary. In particularly preferred embodiments, there is no mismatch at the joint between the connection chartel and the capillary, so that there is zero dead volume in the system when the capillary is fully inserted into the connection channel.
In preferred embodiments, the hybrid apparatus is capable of performing long read-length DNA sequencing of greater than 500, more preferably 800 to 1,000, or even greater than 1,000 bases of DNA sequence in a single run. In preferred embodiments, the apparatus is high-speed (run time of less than 2 hours, more preferably less than 1 hour).
Other embodiments of the invention comprise a rotary scanner for use with the hybrid apparatus (see U.S. Pat. No. 5,483,075, incorporated herein by reference in its entirety).
In additional embodiments, the present invention comprises accessories for automated matrix filling, chip injector cleanup, sample loading and sequencing separation. Design and construction of such accessories may be accomplished by methods well known in the art. In preferred embodiments, the entire system is automated to allow rapid sample throughput with minimal human intervention needed.
In other preferred embodiments, the injector chip is designed to operate with a sample volume of 5 xcexcl or less, more preferably of 0.5 to 2.0 xcexcl, although sample volumes of 0.25 to 0.5 xcexcl or even 0.1 to 0.25 xcexcl are contemplated within the scope of the present invention.
In other embodiments, the chip injector is made of polymer materials, such as polycarbonate, poly(methyl methacrylate) (PMMA), poly(dimethylsiloxane) (PDMA), polystyrene, nitrocellulose, poly(ethylene terephthalate) (PET or Melinex), poly(tetrafluoroethylene) (teflon), etc., using laser ablation, injection molding, casting, or imprinting techniques that are well known in the art.
In certain embodiments, the present invention concerns methods of manufacture of the hybrid apparatus, comprising using a two-mask, or more preferably a three-mask procedure in combination with photolithographic etching of glass wafers to produce a hybrid array with minimal, or more preferably zero dead volume.
In additional embodiments, the present invention concerns methods of use of the hybrid apparatus, comprising using the claimed apparatus for electrophoresis of DNA sequencing products from a Sanger dideoxy reaction. The separated reaction products may be detected and analyzed by standard methods to provide DNA sequence data for long-length, high throughput and high resolution DNA sequencing.
In other embodiments, the present invention concerns methods of use of the hybrid apparatus for separations of other molecules such as peptides, proteins, polysaccharides, lipids and/or oligonucleotides using sieving matrix such as PEO, HEC, agarose, polysaccharides, polyacrylamides, and/or a mixture of those matrices.
In certain embodiments, the present invention concerns apparatus and methods of use of the hybrid device for fluidic communications. Capillaries are used to communicate, for example, between two or more chips, between a chip and an instrument, or between a sample source and a chip. The skilled artisan will realize that these examples are not intended to be limiting, but rather that the capillary array may be used to connect a first chip to any other device, including a second chip. A non-limiting example of the use of the hybrid device for communication between two chips is shown in FIG. 11. Although FIG. 11 shows connections between only two devices, it is contemplated that two, three, four or even more devices could be connected in this way through the use of capillaries. In FIG. 11, a first chip may perform one or more functions such as sample digestion and/or purification. The processed samples are then transferred via one or more capillaries to a second chip. The second chip may perform additional functions such as further sample treatment and/or separation of molecules within the samples. Another non-limiting example would be to use the capillaries to communicate between a chip and an analytical instrument, such as a UVNIS or fluorescence spectrophotometer, a liquid scintillation counter, a charge-coupled device (CCD), a gas chromatograph or a mass spectrometer (MS). Samples may be digested and/or separated on a chip and then delivered through one or more capillaries to an analytical instrument for identification as described in Zhang et al.(1999).